This research is directed at an understanding of the molecular mechanisms involved in the development of resistance to folate analogues in cultured malignant cells. Utilizing various techniques developed in the applicant's laboratories, studies will be pursued to determine the molecular mechanism(s) for the increased synthesis of folate reductase in cultured S-180 mouse fibroblast cells grown in the presence of methotrexate. These studies include analyses of the number of polysomes synthesizing the enzyme, isolation and quantitation of the amount of specific messenger RNA, and determination of the number of gene copies. Following identification of the rate-limiting process, studies will be undertaken to ascertain the mechanisms whereby increased capacity for enzyme synthesis develops as cells are maintained in methotrexate. These studies should provide information on the mechanism of development of resistance in cancer cells to this important class of therapeutic agents.